Glutathione S-transferase (GST) from various arthropods can elicit allergic reactions. In the present study, Per a 5, a GST, was cloned from American cockroach (CR) and expressed in both baculovirus-infected insect cell (iPer a 5) and E. coli expression (bPer a 5) systems. The secondary structures were predicted to be 45.93 and 8.69% of α-helix β-sheets in iPer a 5 and 42.54 and 8.49% of α-helix and β-sheets in bPer a 5, respectively. It is found that 4 out of 16 (25%) sera from American CR allergy patients reacted to both bPer a 9 and iPer a 9 as assessed by ELISA and Western blotting analysis, confirming that Per a 5 is not a major allergen of American CR. Induction of upregulated expression of CD63 and CCR3 on passively sensitized human basophils (sera from American CR allergy patients) by approximately up to 4.5- and 3.2-fold indicates that iPer a 5 and bPer a 5 are functionally active. Recombinant Per a 5 (rPer a 5) should be a useful tool for studying and understanding the role of Per a 5 in CR allergy.